Presently, numerous test devices are available to simply and rapidly analyze body fluids for the presence or absence of a predetermined soluble constituent. For example, tests are available to detect glucose, uric acid or protein in urine; and to detect glucose, triglycerides, potassium ion or cholesterol in blood. Historically, assays of a whole blood sample for a predetermined soluble constituent are the most difficult tests to design.
The cellular components of whole blood, and especially the red blood cells, are the primary interfering substances in assays for a soluble constituent of whole blood. Most simple blood tests are chromogenic, whereby a predetermined soluble constituent of the whole blood interacts with a particular reagent either to form a uniquely-colored compound as a qualitative indication of the presence or absence of the constituent, or to form a colored compound of variable color intensity as a quantitative indication of the presence of the constituent. The deep red color of the whole blood sample interferes with these chromogenic tests, and therefore the highly-colored red blood cells usually are separated from the plasma or serum before the blood sample is assayed for a predetermined soluble constituent.
The presence of red blood cells also can interfere with various nonchromogenic blood assays, whereby the assay results are either inconsistent or, if consistent, are inaccurate. Furthermore, other cellular components, including the white blood cells, also can interfere in standard chromogenic blood assays. Therefore, to achieve a reliable assay for a predetermined soluble constituent of whole blood, it is essential to separate the serum or plasma from the cellular components of whole blood prior to analyzing the whole blood sample for the predetermined soluble constituent.
The assay is further complicated when the predetermined soluble constituent of interest is cholesterol. All cells require cholesterol for growth, but an excess accumulation of cholesterol by the cells can cause various diseases, including atherosclerosis. Therefore, cholesterol is an important analyte because the amount of total serum cholesterol can be correlated to the incidence of atherosclerosis. Accordingly, the assay for cholesterol in serum or plasma is one of the most frequently performed tests in clinical laboratories.
Cholesterol and cholesterol esters are water-insoluble substances, and therefore are carried in the circulatory system by lipoproteins for eventual utilization by the cells. Lipoproteins are complex particles and contain varying amounts of proteins, phospholipids, cholesterol and triglycerides. The lipoproteins in serum are classified by their density. These density-based classes include very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL). Each of these lipoprotein classes carry varying amounts of cholesterol, and a total serum cholesterol assay is a complex average of the amount that each lipoprotein class contributes to the total lipoprotein concentration of the serum.
It is well-known that a specific lipoprotein class, the LDL fraction, is responsible for the accumulation of cholesterol in cells, and is more closely associated with the progression of heart disease, including atherosclerosis. Therefore, the early detection of increased levels of the LDL fraction in blood is of great importance. In contrast, the HDL fraction has been shown to be important in the removal of excess cholesterol from cells. Accordingly, a negative correlation exists between atherosclerosis and HDL cholesterol levels. Additionally, the correlation between atherosclerosis and the level of LDL cholesterol in the blood is higher than a similar correlation between atherosclerosis and total serum cholesterol levels.
High density lipoproteins (HIlL) have been the focus of extensive investigation because of the inverse relationship between HDL cholesterol and the risk of heart infarction. Consequently, if the level of HDL cholesterol in the blood is low, an individual has an increased risk of heart infarction. Thus, the atherosclerosis risk of an individual can be estimated by assaying for HDL cholesterol. The HDL cholesterol assay then is used to calculate the approximate amount of strongly atherogenic LDL cholesterol from the formula: EQU LDL cholesterol=total cholesterol-1/5 total triglycerides-HDL cholesterol.
In order to determine the cholesterol content of the HDL fraction, the other lipoprotein fractions must be removed from the test sample. Four general methods have been developed to separate the lipoprotein fractions. However, each method possesses disadvantages. For example, ultracentrifugation is a common method, but this method is unsuitable for routine laboratory assays because the method requires special equipment, a sensitive manipulative technique and a long separation time that can reach days. Consequently, ultracentrifugation has been restricted to medical research laboratories.
In another method, LDL content is determined by a fractional precipitation reaction utilizing a polyanion, such as heparin sodium or dextran sulfate, and a divalent cation, such as calcium, manganese or magnesium. In this method, the lipoprotein fractions are precipitated in the sequence VLDL, LDL, then HDL, by increasing the concentration of the polyanion. However, this process requires two manipulative steps because the VLDL fraction first is separated in a first precipitation step, then the LDL fraction is precipitated by increasing the concentration of the polyanion. Therefore, the process is impractical and is not amenable to automation.
In the third method, LDL cholesterol is determined from the Friedewald formula. Triglyceride and total cholesterol concentration, including HDL cholesterol content, of the sample is determined by precipitating the VLDL and LDL fractions from the test sample. The amount of LDL cholesterol then is calculated by the Friedewald formula. This method also is laborious and impractical. The fourth method, the electrophoretic separation and polyanion precipitation method, is time-consuming and requires the use of an electrophoresis apparatus and a densitometer for determining LDL cholesterol concentration. Accordingly, not one of the four above-described methods is suitable for the routine assay of HDL cholesterol.
In the assay of a whole blood sample for the amount of cholesterol in the HDL fraction, the cellular components of the whole blood, and the LDL and VLDL fractions of the plasma or serum, are separated from the whole blood sample. Conventionally, the plasma or serum is separated from the cellular material of whole blood by centrifugation. The cellular material collects at the bottom of the centrifuge tube and the supernatant plasma or serum is decanted. Accordingly, the interfering cellular components of whole blood are sufficiently removed such that a substantial background interference is avoided. The supernatant plasma or serum then is subjected to one of the above-described methods of separating the LDL and VLDL fractions from the plasma or serum.
The centrifuge method however has the major disadvantages of requiring a relatively large blood sample, usually from about 0.1 ml to about 5 ml, and requiring a long centrifuge time of approximately 5 to 10 minutes. Furthermore, the centrifuge method requires several manipulative steps. Consequently, a laboratory technician either can contact a potentially-infectious blood sample or can contact laboratory equipment contaminated by the relatively large blood sample, then contract a disease.
Overall, all the above-described separation techniques are best suited for large laboratories that assay a large number of blood samples, and for institutions, such as hospitals, that do not require assay results in a matter of minutes. However, many small laboratories and private medical offices do not have a centrifuge, ultracentrifuge or other such blood, plasma or serum separation devices on site. Therefore, simple chromogenic tests cannot be performed quickly, safely and easily on site, and the whole blood sample is sent to an outside laboratory for efficient and safe separation and assay. As a result, the assay results are available in hours or days as opposed to minutes.
Accordingly, investigators have continually sought a device and method of quickly, safely and easily separating essentially all of the interfering cellular components of whole blood from the plasma or serum such that the identity and concentration of soluble constituents in the plasma or serum are not altered. In addition, for cholesterol assays, a need also exists for a simple and inexpensive device and method of separating the LDL and VLDL fractions from the plasma or serum such that a physician can provide an individual a better estimation of a potential cardiovascular risk than the estimation provided by present day total serum cholesterol assays. Investigators have provided several methods and devices for separating the interfering cellular components and the LDL and VLDL fractions from the plasma or serum. However, each method and device possessed at least one disadvantage that made the method or device inaccurate, cumbersome or impractical in assaying a whole blood sample for the cholesterol present in the HDL fraction of the serum or plasma.
Methods other than centrifugation have been used to separate the cellular components of a small whole blood sample from the serum or plasma. One of the simpler methods, disclosed by Adams et al. in U.S. Pat. No. 3,092,465, used a bibulous, or moisture absorbing, matrix that is impregnated with a chromogenic testing reagent and coated with a semipermeable barrier. The semipermeable barrier screens the cellular components of the whole blood sample and permits passage of the smaller, soluble molecules and ions to contact the chromogenic testing reagent incorporated into the bibulous matrix. In the case of a positive test, the essentially colorless plasma or serum interacts with the chromogenic testing reagent to produce a color in the bibulous matrix. The color is observed by rinsing or wiping the cellular material retained on the semipermeable barrier from the test device. However, the rinsing or wiping technique is cumbersome and laborious, and assay interference is possible if the red blood cells are not completely wiped or rinsed from the semipermeable barrier. In addition, the possibility of technician contact with the potentially-infectious blood sample is high. Mast, in U.S. Pat. No. 3,298,789, disclosed a similar device, wherein a film of ethylcellulose is utilized as the semipermeable barrier. Sodickson, in U.S. Pat. No. 4,059,405, achieved separation of the cellular components from the blood plasma or serum with an ultrafiltration membrane.
Fetter U.S. Pat. Nos. 3,552,925 and 3,552,928 disclosed another method and device to assay whole blood samples for soluble constituents. Fetter described a test device having a bibulous matrix impregnated with a nonvolatile inorganic salt or an amino acid at a first region on the matrix and impregnated with a test reagent at an adjacent second region of the matrix. A whole blood sample is introduced onto the bibulous matrix such that the whole blood first contacts the first region of the bibulous matrix including the inorganic salt or amino acid. The salt or amino acid precipitates the cellular components from the blood, and the plasma or serum then migrates to the test reagent-impregnated second region of the bibulous matrix for a chromogenic interaction with the test reagent.
Another prior art method of separating the cellular components of whole blood from the plasma or serum was disclosed in Vogel et al. U.S. Pat. No. 4,477,575, describing a process and a composition for separating plasma or serum from whole blood using a layer of glass fibers having a defined average diameter and density. However, the amount of plasma or serum that can be separated is limited to at most 50%, and preferably less than 30%, of the absorption volume of the glass fibers. Otherwise, whole blood effectively clogs the glass fiber layer. Therefore, the method requires a high ratio of hydrophobic glass fibers to whole blood volume.
In other prior art methods, the whole blood is diluted before assaying for a predetermined soluble plasma or serum constituent. The dilution of whole blood is burdensome because an extra manipulative step is required, and dilution introduces the possibility of assay error because of an incorrect dilution of the blood sample. The possibility of technician contact with the potentially-infectious blood sample also is increased. For example, German Patent Publication DE-OS 34 41 149 disclosed a method of separating plasma or serum from whole blood by passing the whole blood through a lectin-impregnated matrix that is repeatedly rinsed with a diluent to dilute the plasma or serum before the assay is performed. The use of a lectin or a polymeric amino acid to separate the cellular material from a whole blood sample also is disclosed in European Patent Application No. 84307633.2.
In developing a method and device for separating and assaying small whole blood samples, a primary consideration is the degree of sophistication of the technician performing the assay. Often it is desirable to have relatively untrained personnel perform routine assays and obtain accurate quantitative results. Therefore, it is important that the assay method minimize manipulative steps, be free of possible interferences or contamination, minimize or eliminate the possibility of laboratory personnel physically contacting the blood sample, and provide for easy measurement. For example, among the several possible manipulative steps, the dilution of the whole blood, or the plasma or serum, prior to the actual assay introduces the most probable step for assay error or personal contact with the blood sample. Another common manipulative error is incomplete wiping or rinsing of the cellular components of whole blood from the surface of a device that utilizes a cell-impermeable membrane to separate the cellular components from the plasma or serum of whole blood.
Therefore, a need exists for a method and device to efficiently separate and accurately assay small volumes of whole blood. The method preferably avoids distinct manipulative steps to: 1) separate the cellular components from the plasma or serum, and then 2) to separate a particular component from the plasma or serum prior to the assay. Furthermore, in order to avoid dilution errors, the method preferably allows the assay of undiluted plasma or serum. It also is desirable to provide a blood separation and blood assay method that protects the technician from contact with the blood sample; that avoids the time delays of the present methods; that is independent of the hematocrit value of the blood sample; and that yields accurate and reproducible results.
The ideal method includes withdrawing a whole blood sample in a "noninvasive" amount, such as a pin prick drop, and immediately depositing the undiluted whole blood sample on a test device that separates the cellular components from the undiluted plasma or serum; that separates an undesirable or interfering component from the plasma or serum; and that then assays the undiluted plasma or serum, absent the undesirable component, like the LDL and VLDL fractions, for the presence or concentration of a predetermined soluble constituent, like HDL cholesterol, within minutes. Alternatively, the test device can contact a fresh puncture wound and withdraw a fresh, undiluted blood sample from the wound for analysis. Such a separation and assay method and device allow medical personnel to perform whole blood analyses on a more routine and more confident basis.
Consequently, investigators have attempted to develop test devices that include an element to separate, collect and retain the cellular components of whole blood. Examples of such attempts are disclosed in Vogel et al. U.S. Pat. No. 4,477,575; Rothe et al. U.S. Pat. No. 4,604,265; Kennedy et al. application PCT/US86/02192; Rapkin et al. U.S. Pat. No. 4,678,757; Terminiello et al. U.S. Pat. No. 4,774,192; Stone U.S. Pat. No. 3,607,093; Figueras U.S. Pat. No. 4,144,306; and Pierce et al. U.S. Pat. No. 4,258,001.
Each of the above-identified patents is directed to separating the cellular components of whole blood from the plasma or serum. However, the resulting plasma or serum includes the HDL, the LDL and the VLDL fractions. Therefore, it is still necessary to separate the HDL fraction in the serum or plasma from the LDL and the VLDL fractions in order to accurately determine the amount of HDL cholesterol in the whole blood sample. Some investigators attempted to avoid this second separation by assaying for HDL cholesterol in the plasma or serum indirectly, such as from the difference between the total cholesterol concentration and the sum of cholesterol in the LDL and the VLDL fractions, or by a similar indirect method.
For example, Ziegenhorn et al., in U.S. Pat. No. 4,544,630, disclose a method of assaying for cholesterol in the LDL fraction in the presence of the HDL fraction by a direct enzymatic determination under conditions wherein the LDL fraction interacts with the enzymatic reagents substantially more quickly than the HDL fraction. Sears, in U.S. Pat. Nos. 4,126,416 and 4,190,628, discloses methods and assay kits for LDL cholesterol in blood plasma by separating the LDL cholesterol from the other fractions by agglutinating the LDL fraction with a plant lectin, then detecting the amount of cholesterol in the agglutinated LDL fraction. Sears discloses that either the cholesterol in the LDL fraction can be assayed or, alternatively, the cholesterol in the supernatant liquid, including the HDL and the VLDL fractions can be assayed, then the LDL cholesterol concentration can be determined indirectly.
German Patent Publication DE-OS 31 17 455 discloses a precipitating reagent including phosphotungstic acid and magnesium ions to precipitate the LDL and the VLDL fractions. The cholesterol in the HDL fraction of the supernatant fluid then can be determined. If the total cholesterol concentration of the test sample is known, the HDL cholesterol assay is used in an indirect method of assaying for LDL cholesterol. Not one of the above-identified references teaches or suggests a method or device wherein the test sample is the whole blood, and wherein the cellular components of the whole blood first are separated from the whole blood sample, followed by separation of the LDL and the VLDL fractions from the HDL fraction, in order to assay for HDL cholesterol without a separate precipitating, centrifuging or diluting step. As will be demonstrated more fully hereinafter, the present invention provides a method and a device to assay a small sample of whole blood without time consuming manipulative steps that expose a technician to a potentially-infected test sample and that lead to assay errors.
McGowan, in U.S. Pat. No. 5,118,613, discloses determination of HDL lipoprotein constituents using a whole blood sample. First, the whole blood sample is anticoagulated with a compound like ethylenediaminetetraacetic acid. The LDL and VLDL fractions then are precipitated from the anticoagulated whole blood by magnesium ions and dextran sulfate. The LDL fraction, VLDL fraction and cellular material are separated from the solution by centrifuging, and the supernatant solution, including essentially the entire HDL fraction, is assayed for an HDL constituent, like cholesterol. This method includes the time-consuming and potentially infectious manipulative step of centrifuging.
Other investigators have disclosed devices for assaying blood wherein manipulative diluting and centrifuging steps are eliminated. For example, Kondo et al., in U.S. Pat. No. 4,256,693, disclose a multi-layered device to assay for various serum components, such as cholesterol. The Kondo et al. device includes a filter layer for removing the cellular components of whole blood. The filter layer is positioned over a water-proof layer having at least one small opening. The plasma or serum exiting the filter layer passes through the small opening of the water-proof layer first to contact a spreading layer, then to contact a test area. The analyte of interest then interacts with reagents included in the test area, and a detectable response is observed through the bottom of the device. The device of Kondo et al. cannot measure for HDL cholesterol because all the lipoprotein fractions exit the filter layer to eventually saturate the test area. Accordingly, only total cholesterol is assayed by the device of Kondo et al. device. In addition, and as will be demonstrated more fully hereinafter, the Kondo et al. device does not include a capillary tube, an important feature of the present invention.
Blatt et al., in U.S. Pat. No. 4,761,381, disclose a volume metering device, including a capillary channel, for metering a liquid sample to a reaction chamber. In the Blatt et al. device, the test sample is not filtered. Therefore, in an assay of whole blood, the cellular material interferes in a chromogenic assay for cholesterol because of uneven color development and hematocrit interference in high hematocrit regions. Furthermore, the lipoprotein fractions are not separated, thereby precluding an assay of the whole blood sample for HDL cholesterol.
Hillman et al., in U.S. Pat. No. 4,753,776, disclose a blood separation device that includes a filter and a capillary channel to channel the plasma or serum exiting the filter to a reaction area. Hillman et al. do not teach or suggest further separating the VLDL and LDL fractions from the plasma or serum to provide a method and device to assay for HDL cholesterol.
European Patent Application 168,093 discloses a binder for the LDL fraction. The binder is a sulfated polyvinyl alcohol crosslinked to a water-insoluble substrate. The EPO Application however does not teach separating the cellular components of whole blood from the plasma or serum, followed by separating the lipoprotein fractions in a method to assay for HDL cholesterol. Kerscher et al. U.S. Pat. No. 4,746,605 teaches precipitating the HDL fraction from a test sample, followed by assaying the supernatant liquid for the LDL fraction. In contrast, the present method and device assay the plasma or serum for HDL cholesterol after first separating the cellular components and then separating the LDL fraction and the VLDL fraction from the plasma or serum.
Therefore, because of the disadvantages present in the above-discussed methods and test devices, it is apparent that a simple and effective method of separating the cellular components of whole blood to provide essentially cell-free, unaltered and undiluted plasma or serum, and of separating an undesirable or interfering component of the plasma or serum prior to assaying the plasma or serum for a predetermined analyte, like HDL cholesterol, is needed. Accordingly, the method of the present invention allows the safe, accurate and economical assay of a whole blood sample for HDL cholesterol by utilizing a test device having a separation area including a first zone that separates and retains the cellular components of the whole blood sample from the plasma or serum, and a second zone that separates the LDL fraction and the VLDL fraction from the plasma or serum. The second zone is in intimate contact with, or is in fluid communication with, a test area incorporating the necessary reagents to assay for HDL cholesterol. The plasma or serum exits the separation area in an undiluted form, then migrates to the test area. At the test area, an interaction between the HDL cholesterol and the assay reagents produces a detectable response, such as a color transition, that is free from interferences attributed to highly-colored cellular components and to cholesterol present in the LDL and the VLDL fractions.
The method and device of the present invention allow the assay of whole blood for HDL cholesterol without resorting to a lengthy and expensive extra manipulative step of centrifuging or diluting the test sample. The plasma or serum that saturates the test pad is essentially free of cellular material, the VLDL fraction and the LDL fraction, and is unaltered and undiluted, thereby allowing an accurate and trustworthy assay for HDL cholesterol. The method and device of the present invention also eliminate the disadvantages of hematocrit sensitivity; technique sensitivity due to wiping or rinsing the cellular components from the test device; and disposal of the cellular components.
In accordance with one embodiment of the present invention, after the whole blood sample has passed through the separation area to saturate the test area, a test pad, saturated with undiluted plasma or serum essentially free of the VLDL and the LDL fractions, then is examined for a response to HDL cholesterol by standard dry phase chemistry test strip procedures. In a preferred embodiment, the separation area is not removed from the test device, and a surface of the test pad free from contact with the separation area is examined for a response, such as by examination through a transparent support.
In accordance with another embodiment of the present invention, the separation area and the test area are not in intimate contact, but are in fluid communication by means of a capillary. In this embodiment, the essentially cell-free, LDL-free and VLDL-free plasma or serum migrates from the separation area of the device through a capillary to the test area of the device for an assay of HDL cholesterol in the test sample. The separation area comprises a first zone to remove the cellular components of the whole blood sample and a second zone to remove the VLDL and LDL fractions from the plasma or serum.
In accordance with another important feature of the present invention, the device precludes contact between the technician and the whole blood sample. The blood sample is absorbed into the separation area in such a manner that excess blood sample does not remain on an outside surface of the device. In addition, the technician need not wipe or rinse the cellular components from the device before examination of the device for a response. Consequently, the device essentially eliminates the possibility of contact between the technician and a potentially-infectious blood sample.
As a result of the present invention, the assay of plasma or serum for HDL cholesterol is accurate and reliable because the interferences attributed to the highly-colored cellular components and the LDL and VLDL fractions are essentially eliminated. Prior methods and devices relied upon separating the cellular components from the blood sample by centrifuging, and precipitating and centrifuging the LDL and VLDL fractions from the blood sample. Then, the serum or plasma was assayed for HDL cholesterol by a wet chemistry procedure that was time consuming and required manipulation of the sample and reagents. As will be demonstrated more fully hereinafter, the device of the present invention provides a fast, accurate and economical method of assaying a whole blood sample for HDL cholesterol in the solid or the liquid phase, whereby sample dilutions, reagent and serum manipulation, and technician contact with the blood sample are eliminated.
The separation area utilized in the present invention effectively separates the cellular components from a whole blood sample by utilizing a first separation zone including a filter pad or a portion of a filter pad, that optionally incorporates a relatively small amount of a separating reagent composition, such as an agglutinin, like a lectin; a coagulant, like a thrombin or a thrombin-like compound; or a combination thereof. The optional separating reagent composition incorporated into the first zone of the separation area does not effect the concentration of the lipoprotein fractions in the serum or plasma.
The separation area effectively separates the LDL and VLDL fractions from the serum or plasma by utilizing a second separation zone, including a filter pad or a portion of a filter pad, that incorporates a precipitating reagent composition comprising a polyvalent metal ion, like magnesium ion, and a precipitating compound, like dextran sulfate, therein. Accordingly, undiluted and cell-free serum or plasma including the HDL fraction then migrates to the test area for an assay of HDL cholesterol. Furthermore, the serum or plasma is distributed evenly throughout the entire test pad to provide a homogeneous assay response throughout the test area of the device.
In accordance with an important feature of the present invention, the method and device provide an assay for EDL cholesterol that eliminates wet phase precipitation and centrifugation steps; that eliminates technician handling of the test sample; that eliminates test sample dilution and similar manipulative steps; that utilizes a small blood sample volume; and that eliminates corrections for hematocrit differences between blood samples. Consequently, the test device of the present invention is more economical; separates essentially all of the cellular components and the VLDL and LDL fractions from a whole blood sample; and provides fast, more consistent and more reproducible assays for HDL cholesterol.